This paper describes an integrated microfluidic chip that is capable of rapidly and quantitatively measuring the concentration of a bladder cancer biomarker, apolipoprotein A1, in urine samples. All of the microfluidic components, including the fluid transport system, the micro-valve, and the micro-mixer, were driven by negative pressure, which simplifies the use of the chip and facilitates commercialization. Magnetic beads were used as a solid support for the primary antibody, which captured apolipoprotein A1 in patients' urine. Because of the three-dimensional structure of the magnetic beads, the concentration range of the target that could be detected was as high as 2000 ng ml−1. Because this concentration is 100 times higher than that quantifiable using a 96-well plate with the same enzyme-linked immunosorbent assay (ELISA) kit, the dilution of the patient's urine can be avoided or greatly reduced. The limit of detection was determined to be approximately 10 ng ml−1, which is lower than the cutoff value for diagnosing bladder cancer (11.16 ng ml−1). When the values measured using the microfluidic chip were compared with those measured using conventional ELISA using a 96-well plate for five patients, the deviations were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8%. The entire measurement time is 6-fold faster than that of conventional ELISA. This microfluidic device shows significant potential for point-of-care applications.

Focusing and sorting cells and particles utilizing microfluidic phenomena have been flourishing areas of development in recent years. These processes are largely beneficial in biomedical applications and fundamental studies of cell biology as they provide cost-effective and point-of-care miniaturized diagnostic devices and rare cell enrichment techniques. Due to inherent problems of isolation methods based on the biomarkers and antigens, separation approaches exploiting physical characteristics of cells of interest, such as size, deformability, and electric and magnetic properties, have gained currency in many medical assays. Here, we present an overview of the cell/particle sorting techniques by harnessing intrinsic hydrodynamic effects in microchannels. Our emphasis is on the underlying fluid dynamical mechanisms causing cross stream migration of objects in shear and vortical flows. We also highlight the advantages and drawbacks of each method in terms of throughput, separation efficiency, and cell viability. Finally, we discuss the future research areas for extending the scope of hydrodynamic mechanisms and exploring new physical directions for microfluidic applications.

Microbial fermentation process development is pursuing a high production yield. This requires a high throughput screening and optimization of the microbial strains, which is nowadays commonly achieved by applying slow and labor-intensive submerged cultivation in shake flasks or microtiter plates. These methods are also limited towards end-point measurements, low analytical data output, and control over the fermentation process. These drawbacks could be overcome by means of scaled-down microfluidic microbioreactors (μBR) that allow for online control over cultivation data and automation, hence reducing cost and time. This review goes beyond previous work not only by providing a detailed update on the current μBR fabrication techniques but also the operation and control of μBRs is compared to large scale fermentation reactors.

The instrument described here is an all-electronic dielectrophoresis(DEP) cytometer sensitive to changes in polarizability of single cells. The important novel feature of this work is the differential electrode array that allows independent detection and actuation of single cells within a short section () of the microfluidic channel. DEP actuation modifies the altitude of the cells flowing between two altitude detection sites in proportion to cellpolarizability; changes in altitude smaller than 0.25 μm can be detected electronically. Analysis of individual experimental signatures allows us to make a simple connection between the Clausius-Mossotti factor (CMF) and the amount of vertical cell deflection during actuation. This results in an all-electronic, label-free differential detector that monitors changes in physiological properties of the living cells and can be fully automated and miniaturized in order to be used in various online and offline probes and point-of-care medical applications. High sensitivity of the DEP cytometer facilitates observations of delicate changes in cell polarization that occur at the onset of apoptosis. We illustrate the application of this concept on a population of Chinese hamster ovary (CHO) cells that were followed in their rapid transition from a healthy viable to an early apoptotic state. DEP cytometer viability estimates closely match an Annexin V assay (an early apoptosis marker) on the same population of cells.

Using Monte Carlo simulations of a touching-bead model of double-stranded DNA, we show that DNA extension is enhanced in isosceles triangular nanochannels (relative to a circular nanochannel of the same effective size) due to entropic depletion in the channel corners. The extent of the enhanced extension depends non-monotonically on both the accessible area of the nanochannel and the apex angle of the triangle. We also develop a metric to quantify the extent of entropic depletion, thereby collapsing the extension data for circular, square, and various triangular nanochannels onto a single master curve for channel sizes in the transition between the Odijk and de Gennes regimes.

This paper describes an integrated microfluidic chip that is capable of rapidly and quantitatively measuring the concentration of a bladder cancer biomarker, apolipoprotein A1, in urine samples. All of the microfluidic components, including the fluid transport system, the micro-valve, and the micro-mixer, were driven by negative pressure, which simplifies the use of the chip and facilitates commercialization. Magnetic beads were used as a solid support for the primary antibody, which captured apolipoprotein A1 in patients' urine. Because of the three-dimensional structure of the magnetic beads, the concentration range of the target that could be detected was as high as 2000 ng ml−1. Because this concentration is 100 times higher than that quantifiable using a 96-well plate with the same enzyme-linked immunosorbent assay (ELISA) kit, the dilution of the patient's urine can be avoided or greatly reduced. The limit of detection was determined to be approximately 10 ng ml−1, which is lower than the cutoff value for diagnosing bladder cancer (11.16 ng ml−1). When the values measured using the microfluidic chip were compared with those measured using conventional ELISA using a 96-well plate for five patients, the deviations were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8%. The entire measurement time is 6-fold faster than that of conventional ELISA. This microfluidic device shows significant potential for point-of-care applications.

In this work, we conduct a computational study on the loading of cryoprotective agents into cells in preparation for cryopreservation. The advantages of microfluidics in cryopreserving cells include control of fluid flow parameters for reliable cryoprotectant loading and reproducible streamlined processing of samples. A 0.25 m long, three inlet T-junction microchannel serves as an idealized environment for this process. The flow field and concentration distribution are determined from a computational fluid dynamics study and cells are tracked as inert particles in a Lagrangian frame. These particles are not confined to streamlines but can migrate laterally due to the Segre-Sildeberg effect for particles in a shear flow. During this tracking, the local concentration field surrounding the cell is monitored. This data are used as input into the Kedem-Katchalsky equations to numerically study passive solute transport across the cell membrane. As a result of the laminar flow, each cell has a unique pathline in the flow field resulting in different residence times and a unique external concentration field along its path. However, in most previous studies, the effect of a spatially varying concentration field on the transport across the cell membrane is ignored. The dynamics of this process are investigated for a population of cells released from the inlet. Using dimensional analysis, we find a governing parameter α, which is the ratio of the time scale for membranetransport to the average residence time in the channel. For , cryoprotectant loading is completed to within 5% of the target concentration for all of the cells. However, for , we find the population of cells does not achieve complete loading and there is a distribution of intracellular cryoprotective agent concentration amongst the population. Further increasing α beyond a value of 2 leads to negligible cryoprotectant loading. These simulations on populations of cells may lead to improved microfluidic cryopreservation protocols where more consistent cryoprotective agent loading and freezing can be achieved, thus increasing cell survival.

Cytokines are small proteins secreted by leukocytes in blood in response to infections, thus offering valuable diagnostic information. Given that the same cytokines may be produced by different leukocyte subsets in blood, it is beneficial to connect production of cytokines to specific cell types. In this paper, we describe integration of antibody (Ab) microarrays into a microfluidic device to enable enhanced cytokine detection. The Ab arrays contain spots specific to cell-surface antigens as well as anti-cytokine detection spots. Infusion of blood into a microfluidic device results in the capture of specific leukocytes (CD4 T-cells) and is followed by detection of secreted cytokines on the neighboring Ab spots using sandwich immunoassay. The enhancement of cytokine signal comes from leveraging the concept of reconfigurable microfluidics. A three layer polydimethylsiloxanemicrofluidic device is fabricated so as to contain six microchambers (1 mm × 1 mm × 30 μm) in the ceiling of the device. Once the T-cell capture is complete, the device is reconfigured by withdrawing liquid from the channel, causing the chambers to collapse onto Ab arrays and enclose cell/anti-cytokine spots within a 30 nl volume. In a set of proof-of-concept experiments, we demonstrate that ∼90% pure CD4 T-cells can be captured inside the device and that signals for three important T-cell secreted cytokines, tissue necrosis factor-alpha, interferon-gamma, and interleukin-2, may be enhanced by 2 to 3 folds through the use of reconfigurable microfluidics.

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic,electroosmotic (EO), electrophoretic(EP), and dielectrophoretic(DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and ACDEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.

Acoustic trapping of minute bead amounts against fluid flow allows for easy automation of multiple assay steps, using a convenient aspirate/dispense format. Here, a method based on acoustic trapping that allows sample preparation for immuno-matrix-assisted laser desorption/ionization mass spectrometry using only half a million 2.8 μm antibody covered beads is presented. The acoustic trapping is done in 200 × 2000 μm2 glass capillaries and provides highly efficient binding and washing conditions, as shown by complete removal of detergents and sample processing times of 5-10 min. The versatility of the method is demonstrated using an antibody against Angiotensin I (Ang I), a peptide hormone involved in hypotension. Using this model system, the acoustic trapping was efficient in enriching Angiotensin at 400 pM spiked in plasma samples.

We introduce a novel type of droplet generator that produces droplets of a volume set by the geometry of the droplet generator and not by the flow rates of the liquids. The generator consists of a classic T-junction with a bypass channel. This bypass directs the continuous fluid around the formingdroplets, so that they can fill the space between the inlet of the dispersed phase and the exit of the bypass without breaking. Once filled, the dispersed phase blocks the exit of the bypass and is squeezed by the continuous fluid and broken off from the junction. We demonstrate the fixed-volume droplet generator for (i) the formation of monodisperse droplets from a source of varying flow rates, (ii) the formation of monodisperse droplets containing a gradation of solute concentration, and (iii) the parallel production of monodisperse droplets.

The capability of the AC dielectrophoresis(DEP) for on-chip capture and chaining of microalgae suspended in freshwaters was evaluated. The effects of freshwater composition as well as the electric field voltage, frequency, and duration, on the dielectrophoretic response of microalga Chlamydomonas reinhardtii were characterized systematically. Highest efficiency of cell alignment in one-dimensional arrays, determined by the percentage of cells in chain and the chain length, was obtained at AC-field of 20 V mm−1 and 1 kHz applied for 600 s. The DEP response and cell alignment of C. reinhardtii in water sampled from lake, pond, and river, as well as model media were affected by the chemical composition of the media. In the model media, the efficiency of DEP chaining was negatively correlated to the conductivity of the cell suspensions, being higher in suspensions with low conductivity. The cells suspended in freshwaters, however, showed anomalously high chaining at long exposure times. High concentrations of nitrate and dissolved organic matter decrease cell chaining efficiency, while phosphate and citrate concentrations increase it and favor formation of longer chains. Importantly, the application of AC-field had no effect on algal autofluorescence, cell membrane damage, or oxidative stress damages in C. reinhardtii.

This study proposes a capillary dielectrophoretic chip to separate blood cells from a drop of whole blood (approximately 1 μl) sample using negative dielectrophoretic force. The separating efficiency was evaluated by analyzing the image before and after dielectrophoretic force manipulation. Blood samples with various hematocrits (10%–60%) were tested with varied separating voltages and chip designs. In this study, a chip with 50 μm gap design achieved a separation efficiency of approximately 90% within 30 s when the hematocrit was in the range of 10%–50%. Furthermore, glucose concentration was electrochemically measured by separating electrodes following manipulation. The current response increased significantly (8.8-fold) after blood cell separation, which was attributed not only to the blood cell separation but also to sample disturbance by the dielectrophoretic force.

We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cellvelocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamiclubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data.

Nanofluidics has a unique property that ionic conductance across a nanometer-sized confined space is strongly affected by the space surface charge density, which can be utilized to construct electrical read-out biosensor. Based on this principle, this work demonstrated a novel proteinsensor along with a sandwich signal enhancement approach. Nanoparticles with designed aptamer onside are assembled in a suspended micropore to form a 3-dimensional network of nanometer-sized interstices, named as nanofluidic crystal hereafter, as the basic sensing unit. Proteins captured by aptamers will change the surface charge density of nanoparticles and thereby can be detected by monitoring the ionic conductance across this nanofluidic crystal. Another aptamer can further enlarge the variations of the surface charge density by forming a sandwich structure (capturing aptamer/protein/signal enhancement aptamer) and the read-out conductance as well. The preliminary experimental results indicated that human α-thrombin was successfully detected by the corresponding aptamer modified nanofluidic crystal with the limit of detection of 5 nM (0.18 μg/ml) and the read-out signal was enhanced up to 3 folds by using another thrombin aptamer. Being easy to graft probe, facile and low-cost to prepare the nano-device, and having an electrical read-out, the present nanofluidic crystal scheme is a promising and universal strategy for protein sensing.

Thanks to its unique features at the nanoscale, nanofluidics, the study and application of fluid flow in nanochannels/nanopores with at least one characteristic size smaller than 100 nm, has enabled the occurrence of many interesting transport phenomena and has shown great potential in both bio- and energy-related fields. The unprecedented growth of this research field is apparently attributed to the rapid development of micro/nanofabrication techniques. In this review, we summarize recent activities and achievements of nanofabrication for nanofluidic devices, especially those reported in the past four years. Three major nanofabrication strategies, including nanolithography, microelectromechanical system based techniques, and methods using various nanomaterials, are introduced with specific fabrication approaches. Other unconventional fabrication attempts which utilize special polymer properties, various microfabrication failure mechanisms, and macro/microscale machining techniques are also presented. Based on these fabrication techniques, an inclusive guideline for materials and processes selection in the preparation of nanofluidic devices is provided. Finally, technical challenges along with possible opportunities in the present nanofabrication for nanofluidic study are discussed.

Polymer-based microneedles have drawn much attention in the transdermal drug delivery resulting from their flexibility and biocompatibility. Traditional fabrication approach deploys various kinds of molds to create sharp tips at the end of needles for the penetration purpose. This approach is usually time-consuming and expensive. In this study, we developed an innovative fabrication process to make biocompatible SU-8 microtubes integrated with biodissolvable maltose tips as novel microneedles for the transdermal drug delivery applications. These microneedles can easily penetrate the skin's outer barrier represented by the stratum corneum (SC) layer. The drug deliverydevice of mironeedles array with 1000 μm spacing between adjacent microneedles is proven to be able to penetrate porcine cadaver skins successfully. The maximum loading force on the individual microneedle can be as large as 7.36 ± 0.48N. After 9 min of the penetration, all the maltose tips are dissolved in the tissue. Drugs can be further delivered via these open biocompatible SU-8 microtubes in a continuous flow manner. The permeation patterns caused by the solution containing Rhodamine 110 at different depths from skinsurface were characterized via a confocal microscope. It shows successful implementation of the microneedle function for fabricated devices.

Surface analysis is critical for the validation of microfluidicsurface modifications for biology, chemistry, and physics applications. However, until now quantitative analytical methods have mostly been focused on open surfaces. Here, we present a new fluorescenceimaging method to directly measure the surface coverage of functional groups inside assembled microchannels over a wide dynamic range. A key advance of our work is the elimination of self-quenching to obtain a linear signal even with a high density of functional groups. This method is applied to image the density and monitor the stability of vapor deposited silane layers in bonded silicon/glass micro- and nanochannels.